Volume 133, Issue 1, Pages 219-231 (July 2007) Bidirectional Signals Transduced by TOPK-ERK Interaction Increase Tumorigenesis of HCT116 Colorectal Cancer Cells  Feng Zhu, Tatyana A. Zykova, Bong Seok Kang, Zhe Wang, Mara C. Ebeling, Yasuhito Abe, Wei–Ya Ma, Ann M. Bode, Zigang Dong  Gastroenterology  Volume 133, Issue 1, Pages 219-231 (July 2007) DOI: 10.1053/j.gastro.2007.04.048 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 TOPK is overexpressed in colorectal cancer patients and colorectal cancer cell lines. (A) Expression of TOPK in 9 different cell lines. (B) Immunohistochemical examination for the expression of TOPK in 49 cases of human colorectal cancer tissues and matching normal colorectal tissue. The table summarizes the results of the immunohistochemical examination. Pictures from 1 representative case are shown below the table. The 3 scale bars from top to bottom in each group correspond to 250, 50, and 25 μm, respectively. (C) High expression of TOPK in 30 colorectal cancer cell lines. Cell lines are listed in the table, and the representative expression of TOPK in HCT116 cells is shown below the table. Gastroenterology 2007 133, 219-231DOI: (10.1053/j.gastro.2007.04.048) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 TOPK transforms mouse epidermal JB6 Cl41 cells in vitro and in vivo. (A) Growth curves of vector control cells (JB6-Mock) and TOPK-overexpressing cells (JB6-TOPK). Inset shows verification of the cell lines identified by Western blot. Data are represented as mean ± SD of 3 experiments. The asterisk indicates a significant increase (P < .001) in cell number in JB6-TOPK cells compared with JB6-Mock cells. (B) TOPK can transform JB6 Cl41 cells in vitro as illustrated by growth of TOPK transformed cells in soft agar. Photomicrograph of representative colony formation in soft agar of vector control cells (JB6-Mock) compared with TOPK-overexpressing cells (JB6-TOPK) is shown. (C) TOPK can transform JB6 Cl41 cells in vivo. Representative picture from each group of mice injected with vector control cells (JB6-Mock) or TOPK-overexpressing cells (JB6-TOPK) is shown. The arrows indicate the injection site and tumor growth. (D) Representative H&E staining obtained from a tumor in the JB6-TOPK group. The scale bars from left to right correspond to 250 and 25 μm, respectively. (E) Final average tumor weight and tumor growth curve of mice injected with JB6-Mock or JB6-TOPK cells. Data are expressed as means ± SE of 8 mice in each group. No tumors were formed in mice injected with JB6-Mock cells. Gastroenterology 2007 133, 219-231DOI: (10.1053/j.gastro.2007.04.048) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Knockdown of TOPK in HCT116 reduces tumorigenic properties in vitro and in vivo. (A) Growth curves of HCT116-siMock and HCT116-siTOPK cell lines. Inset shows verification of the cell lines identified by Western blot. Data are represented as mean ± SD of 3 experiments. The asterisk indicates a significant increase (P < .001) in cell number in HCT116-siMock cells compared with HCT116-siTOPK cells. (B) Knockdown of TOPK reduces tumorigenic properties of colorectal cancer cell line HCT116 in vitro. Representative photomicrograph of colony formation in soft agar of HCT116-siMock cells compared with HCT116-siTOPK cells is shown. (C) Knockdown of TOPK reduces tumorigenic properties of colorectal cancer cell line HCT116 in vivo. Representative picture of a mouse from each group injected with HCT116-siMock or HCT116-siTOPK cells is shown. The arrows indicate the injection site and tumor location. Tumors dissected from each group are shown below. (D) Final average tumor weight and tumor growth curve of mice injected with HCT116-siMock or HCT116-siTOPK cells. Data are expressed as means ± SE of 11 mice in each group. The asterisk indicates a significant increase in tumor weight and size in HCT116-siMock-injected mice compared with HCT116-siTOPK injected mice (P < .02). Gastroenterology 2007 133, 219-231DOI: (10.1053/j.gastro.2007.04.048) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 TOPK phosphorylates ERK2 in vitro. (A) Alignment of the amino acids sequences between MEK1 and TOPK near the region of MEK1 active sites: S217 and S221. The upper diagram shows the different residues between MEK1 and TOPK near the MEK1 active sites. (B) Active TOPK phosphorylates inactive ERK2 in vitro in the presence of γ-32P-ATP as visualized by autoradiography (left panel). TOPK phosphorylation of inactive p38 was used as a positive control (right panel). (C) Active TOPK phosphorylates inactive ERK2 in vitro as detected by Western blot using anti-phosphor-ERKs or anti-phosphor-TOPK (left panel). TOPK phosphorylation of inactive p38 was used as a positive control (right panel). (D) Active TOPK phosphorylates His-ERK2 in vitro in the presence of γ-32P-ATP as visualized by autoradiography. Gastroenterology 2007 133, 219-231DOI: (10.1053/j.gastro.2007.04.048) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 ERK2 phosphorylates TOPK in vitro. (A) Active ERK2 phosphorylates His-TOPK in vitro in the presence of γ-32P-ATP as visualized by autoradiography. (B) Active ERK2 phosphorylates His-TOPK in vitro as detected by Western blot using anti-phosphor-TOPK. Silver-stained Figure below verifies equal sample loading for the kinase assay. (C) TOPK phosphorylation increases in a dose-dependent manner corresponding to the increased amount of inactive ERK2. Phosphorylation of TOPK or ERKs and total ERK2 or TOPK were detected by Western blot. (D) Inactive ERK2 cannot phosphorylate ELK1. Gastroenterology 2007 133, 219-231DOI: (10.1053/j.gastro.2007.04.048) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 The feedback loop between TOPK and ERK2 correlates with increased kinase activity. (A) TOPK binds with ERK2 in HEK 293 cells after transient transfection as indicated. pcDNA3-HA-TOPK and pcDNA3-V5-ERK2 were cotransfected into HEK 293 cells, immunoprecipitated with a HA or V5 antibody, and then probed with V5 or HA, respectively. (B) TOPK binds with ERK2 in HCT116 cells. Endogenous ERK2 was immunoprecipitated from HCT116 cells and then probed with anti-TOPK. (C) TOPK promotes phosphorylation of ERK2 in HEK 293 cells after transient transfection as indicated. HEK 293 cells were transfected with plasmids as indicated and then serum starved for 24 hours and stimulated with 20 ng/mL EGF for 15 minutes. The samples were then analyzed by Western blot. (D) Phosphorylation of ERK2 by TOPK enhances kinase activity of ERK2. The samples in C were subjected to immunoprecipitation using the V5 antibody, and then a kinase assay was performed with GST-ELK1 as substrate. (E) ERK2 promotes phosphorylation of TOPK in HEK 293 cells after transient transfection as indicated. HEK 293 cells were transfected with plasmids as indicated and then serum starved for 24 hours and stimulated with 20 ng/mL EGF for 15 minutes. The samples were then analyzed by Western blot. (F) Phosphorylation of TOPK by ERK2 enhances kinase activity of TOPK. The samples in C were subjected to immunoprecipitation using the HA antibody, and then a kinase assay was performed with GST-ERK2 as substrate. (G) TOPK promotes phosphorylation of endogenous ERKs in HEK 293 cells in a dose-dependent manner after EGF treatment for 15 minutes. Increasing amounts of HA-TOPK were transiently transfected into HEK 293 cells, and phosphorylation of ERKs was detected by Western blot. (H) ERK2 promotes phosphorylation of endogenous TOPK in HEK 293 cells in a dose-dependent manner after EGF treatment for 15 minutes. Increasing amounts of V5-ERK2 were transiently transfected into HEK 293 cells, and phosphorylation of TOPK was detected by Western blot. (I) Phosphorylation of ERKs was increased in JB6-TOPK cells after EGF treatment for 15 minutes. Gastroenterology 2007 133, 219-231DOI: (10.1053/j.gastro.2007.04.048) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 The feedback loop between TOPK and ERK2 correlates with increased kinase activity. (A) TOPK binds with ERK2 in HEK 293 cells after transient transfection as indicated. pcDNA3-HA-TOPK and pcDNA3-V5-ERK2 were cotransfected into HEK 293 cells, immunoprecipitated with a HA or V5 antibody, and then probed with V5 or HA, respectively. (B) TOPK binds with ERK2 in HCT116 cells. Endogenous ERK2 was immunoprecipitated from HCT116 cells and then probed with anti-TOPK. (C) TOPK promotes phosphorylation of ERK2 in HEK 293 cells after transient transfection as indicated. HEK 293 cells were transfected with plasmids as indicated and then serum starved for 24 hours and stimulated with 20 ng/mL EGF for 15 minutes. The samples were then analyzed by Western blot. (D) Phosphorylation of ERK2 by TOPK enhances kinase activity of ERK2. The samples in C were subjected to immunoprecipitation using the V5 antibody, and then a kinase assay was performed with GST-ELK1 as substrate. (E) ERK2 promotes phosphorylation of TOPK in HEK 293 cells after transient transfection as indicated. HEK 293 cells were transfected with plasmids as indicated and then serum starved for 24 hours and stimulated with 20 ng/mL EGF for 15 minutes. The samples were then analyzed by Western blot. (F) Phosphorylation of TOPK by ERK2 enhances kinase activity of TOPK. The samples in C were subjected to immunoprecipitation using the HA antibody, and then a kinase assay was performed with GST-ERK2 as substrate. (G) TOPK promotes phosphorylation of endogenous ERKs in HEK 293 cells in a dose-dependent manner after EGF treatment for 15 minutes. Increasing amounts of HA-TOPK were transiently transfected into HEK 293 cells, and phosphorylation of ERKs was detected by Western blot. (H) ERK2 promotes phosphorylation of endogenous TOPK in HEK 293 cells in a dose-dependent manner after EGF treatment for 15 minutes. Increasing amounts of V5-ERK2 were transiently transfected into HEK 293 cells, and phosphorylation of TOPK was detected by Western blot. (I) Phosphorylation of ERKs was increased in JB6-TOPK cells after EGF treatment for 15 minutes. Gastroenterology 2007 133, 219-231DOI: (10.1053/j.gastro.2007.04.048) Copyright © 2007 AGA Institute Terms and Conditions

Figure 7 The positive feedback loop between TOPK and ERK2 exists in HCT116 cells. (A) EGF induces phosphorylation of TOPK in HCT116 cells. (B) Phosphorylation of ERKs decreases with knockdown of TOPK in HCT116 cells. (C) Phosphorylation of TOPK decreases with knockdown of ERK2 in HCT116 cells. (D) Downstream signaling pathways of ERKs are affected with knockdown of TOPK. The levels of phosphorylation of c-MYC, c-FOS, CREB, and ELK1 were detected in HCT116-siTOPK cells or HCT116-siMock at 15 minutes after treatment with 20 ng/mL EGF. The asterisk denotes nonspecific signals. Gastroenterology 2007 133, 219-231DOI: (10.1053/j.gastro.2007.04.048) Copyright © 2007 AGA Institute Terms and Conditions